The present invention relates to chemically modified mutant proteins having modified glycosylation patterns with respect to a precursor protein from which they are derived. In particular, the present invention relates to a chemically modified mutant protein including a cysteine residue substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate. The present invention also relates to a method of producing the chemically modified mutant proteins and a glycosylated methanethiosulfonate. Another aspect of the present invention is a method of modifying the functional characteristics of a protein by reacting the protein with a glycosylated methanethiosulfonate reagent. The present invention also relates to methods of determining the structure-function relationships of chemically modified mutant proteins.
Modifying enzyme properties by site-directed mutagenesis has been limited to natural amino acid replacements, although molecular biological strategies for overcoming this restriction have recently been derived (Cornish et al., Angew. Chem., Int. Ed. Engl., 34:621-633 (1995)). However, the latter procedures are difficult to apply in most laboratories. In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity.
Changing enzyme properties by chemical modification has been explored previously, with the first report being in 1966 by the groups of Bender (Polgar et al., J. Am. Chem. Soc., 88:3153-3154 (1966)) and Koshland (Neet et al., Proc. Natl. Acad. Sci. USA, 56:1606-1611 (1966)), who created a thiolsubtilisin by chemical transformation (CH2OHxe2x86x92CH2SH) of the active site serine residue of subtilisin BPN"" to cysteine. Interest in chemically produced artificial enzymes, including some with synthetic potential, was renewed by Wu (Wu et al., J. Am. Chem. Soc., 111:4514-4515 (1989); Bell et al., Biochemistry, 32:3754-3762 (1993)) and Peterson (Peterson et al., Biochemistry, 34:6616-6620 (1995)), and, more recently, Suckling (Suckling et al., Bioorg. Med. Chem. Lett., 3:531-534 (1993)).
Enzymes are now widely accepted as useful catalysts in organic synthesis. However, natural, wild-type, enzymes can never hope to accept all structures of synthetic chemical interest, nor always be transformed stereospecifically into the desired enantiomerically pure materials needed for synthesis. This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis. The generation of new specificities or activities obtained by chemical modification of enzymes has intrigued chemists for many years and continues to do so.
U.S. Pat. No. 5,208,158 to Bech et al. (xe2x80x9cBechxe2x80x9d) describes chemically modified detergent enzymes where one or more methionines have been mutated into cysteines. The cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with C1-6 alkyl.
Although Bech has described altering the oxidative stability of an enzyme through mutagenesis and chemical modification, it would also be desirable to develop one or more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, pH activity profile, and surface binding properties for use in, for example, detergents or organic synthesis. In particular, enzymes, such as subtilisins, tailored for peptide synthesis would be desirable. Enzymes useful for peptide synthesis have high esterase and low amidase activities. Generally, subtilisins do not meet these requirements and the improvement of the esterase to amidase selectivities of subtilisins would be desirable. However, previous attempts to tailor enzymes for peptide synthesis by lowering amidase activity have generally resulted in dramatic decreases in both esterase and amidase activities. Previous strategies for lowering the amidase activity include the use of water-miscible organic solvents (Barbas et al., J. Am. Chem. Soc., 110:5162-5166 (1988); Wong et al., J. Am. Chem. Soc., 112:945-953 (1990); and Sears et al., Biotechnol. Prog., 12:423-433 (1996)) and site-directed mutagenesis (Abrahamsen et al., Biochemistry, 30:4151-4159 (1991); Bonneau et al., xe2x80x9cAlteration of the Specificity of Subtilisin BPN"" by Site-Directed Mutagenesis in its S1 and S1xe2x80x2 Binding-Sites,xe2x80x9d J. Am. Chem. Soc., 113:1026-1030 (1991); and Graycar et al., Ann. N. Y. Acad. Sci., 67:71-79 (1992)). However, while the ratios of esterase-to-amidase activities were improved by these approaches, the absolute esterase activities were lowered concomitantly. Abrahamsen et al., Biochemistry, 30:4151-4159 (1991). Chemical modification techniques (Neet et al., Proc. Nat. Acad. Sci., 56:1606 (1966); Polgar et al., J. Am. Chem. Soc., 88:3153-3154 (1966); Wu et al., J. Am. Chem. Soc., 111:4514-4515 (1989); and West et al., J. Am. Chem. Soc., 112:5313-5320 (1990)), which permit the incorporation of unnatural amino acid moieties, have also been applied to improve esterase to amidase selectivity of subtilisins. For example, chemical conversion of the catalytic triad serine (Ser221) of subtilisin to cysteine (Neet et al., Proc. Nat. Acad. Sci., 56:1606 (1966); Polgar et al., J. Am. Chem. Soc., 88:3153-3154 (1966); and Nakatsukaet al., J. Am. Chem. Soc., 109:3808-3810 (1987)) or to selenocysteine (Wu et al., J. Am. Chem. Soc., 111:4514-4515 (1989)), and methylation of the catalytic triad histidine (His57) of chymotrypsin (West et al., J. Am. Chem. Soc., 112:5313-5320 (1990)), effected substantial improvement in esterase-to-amidase selectivities. Unfortunately however, these modifications were again accompanied by 50- to 1000-fold decreases in absolute esterase activity.
Surface glycoproteins act as markers in cell-cell communication events that determine microbial virulence (Sharon et al., Essays Biochem., 30:59-75 (1995)), inflammation (Lasky, Annu. Rev. Biochem., 64:113-139 (1995); Weis et al., Annu. Rev. Biochem., 65:441-473 (1996)), and host immune responses (Varki, Glycobiol., 3:97-130 (1993); Dwek, Chem. Rev., 96:683-720 (1996)). In addition, the correct glycosylation of proteins is critical to their expression and folding (Helenius, Mol. Biol. Cell, 5:253-265 (1994)) and increases their thermal and proteolytic stability (Opdenakker et al., FASEB J., 7:1330-1337(1993)). Glycoproteins occur naturally in a number of forms (glycoforms) (Rademacher et al., Annu. Rev. Biochem., 57:785-838 (1988)) that possess the same peptide backbone, but differ in both the nature and site of glycosylation. The differences exhibited (Rademacher et al., Annu. Rev. Biochem., 57:785-838 (1988); Parekh et al., Biochem., 28:7670-7679 (1989); Knight, Biotechnol., 7:35-40 (1989)) by each component within these microheterogeneous mixtures present regulatory difficulties (Liu, Trends Biotechnol., 10:114-120 (1992); Bill et al., Chem. Biol., 3:145-149 (1996)) and problems in determining exact function. To explore these key properties, there is a pressing need for methods that will not only allow the preparation of pure glycosylated proteins, but will also allow the preparation of non-natural variants for the determination of structure-function relationships, such as structure-activity relationships (SARs). The few studies that have compared single glycoforms successfully have required abundant sources and extensive chromatographic separation (Rudd et al., Biochem., 33:17-22 (1994)). Neoglycoproteins (Krantz et al., Biochem., 15:3963-3968 (1976)), formed via unnatural linkages between sugars and proteins, provide an invaluable alternative source of carbohydrate-protein conjugates (For reviews see Stowell et al., Adv. Carbohydr. Chem. Biochem., 37:225-281 (1980); Neoglycoconjugates: Preparation and Applications, Lee et al., Eds., Academic Press, London (1994); Abelson et al., Methods Enzymol., 242: (1994); Lee et al., Methods Enzymol., 247: (1994); Bovin et al., Chem. Soc. Rev., 24:413-421 (1995)). In particular, chemical glycosylation allows control of the glycan structure and the nature of the sugar-protein bond. However, despite these advantages, existing methods for their preparation (Stowell et al., Adv. Carbohydr. Chem. Biochem., 37:225-281 (1980)) typically generate mixtures. In addition, these techniques may alter the overall charge of the protein (Lemieux et al., J. Am. Chem. Soc., 97:4076-4083 (1975); Kobayashi et al., Methods Enzymol., 247:409-418 (1994)) or destroy the cyclic nature of glycans introduced (Gray, Arch. Biochem. Biophvs., 163:426-428 (1974)). For example, the reductive amination of lactose with bovine serum albumin (BSA) caused indiscriminate modification of lysine residues through the formation of acyclic amines introduced (Gray, Arch. Biochem. Biophys., 163:426-428 (1974)). Advances in the site-specific glycosylation of BSA have been made (Davis et al., Tetrahedron Lett., 32:6793-6796 (1991); Wong et al., Biochem. J., 300:843-850 (1994); Macindoe et al., J. Chem. Soc., Chem. Commun., 847-848 (1998)). However, these methods rely upon modification of an existing cysteine in BSA and, as such, allow no flexibility in the choice of glycosylation site. Glycoproteins occur naturally as complex mixtures of differently glycosylated forms which are difficult to separate. To explore their properties, there is a need for homogenous sources of carbohydrate-protein conjugates. Existing methods typically generate product protein mixtures of poorly characterized composition, with little or no control over the site or level of glycosylation.
The present invention is directed to overcoming these deficiencies.
It is an object of the present invention to provide for novel glycosylated proteins.
It is a further object of the invention to provide for novel glycosylated proteins which have modified or improved functional characteristics.
It is a further object of the invention to provide for a method of producing glycosylated proteins which have well defined properties, for example, by having predetermined glycosylation patterns.
According to the present invention, a method is provided wherein the glycosylation pattern of a protein is modified in a predictable and repeatable manner. Generally, the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate.
Thus, in one composition aspect of the present invention, a chemically modified mutant protein is provided, wherein said mutant protein differs from a precursor protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by reacting said cysteine residue with a glycosylated thiosulfonate. Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably a methanethiosulfonate.
In a method aspect of the present invention, a method of producing a chemically modified mutant protein is provided comprising the steps of: (a) providing a precursor protein; (b) substituting an amino acid residue other than cysteine in said precursor protein with a cysteine; (c) reacting said substituted cysteine with a glycosylated thiosulfonate, said glycosylated thiosulfonate comprising a carbohydrate moiety; and (d) obtaining a modified glycosylated protein wherein said substituted cysteine comprises a carbohydrate moiety attached thereto. Preferably, the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate. Also preferably, the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein.
The present invention also relates to novel glycosylated thiosulfonates. In a preferred embodiment, the glycosylated thiosulfonate is a methanethiosulfonate. In a most preferred embodiment, the glycosylated methanethiosulfonate comprises a chemical structure including: 
where R comprises -xcex2-Glc, -Et-xcex2-Gal, -Et-xcex2-Glc, -Et-xcex1-Glc, -Et-xcex1-Man, -Et-Lac, -xcex2-Glc(Ac)2, -xcex2-Glc(Ac)3, -xcex2-Glc(Ac)4, -Et-xcex1-Glc(Ac)2, -Et-xcex1-Glc(Ac)3, -Et-xcex1-Glc(Ac)4, -Et-xcex2-Glc(Ac)2, -Et-xcex2-Glc(Ac)3, -Et-xcex2-Glc(Ac)4, -Et-xcex1-Man(Ac)3, -Et-xcex1-Man(Ac)4, -Et-xcex2-Gal(Ac)3, -Et-xcex2-Gal(Ac)4, -Et-Lac(Ac)5, -Et-Lac(Ac)6, or -Et-Lac(Ac)7.
Another aspect of the present invention is a method of modifying the functional characteristics of a protein including reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein. Accordingly, the present invention provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein.
The present invention also relates to methods of determining the structure-function relationships of chemically modified mutant proteins. One method includes providing first and second chemically modified mutant proteins of the present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins. Another method involves providing first and second chemically modified mutant proteins of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting said cysteine residue with a glycosylated thiosulfonate, evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
The chemically modified mutant proteins of the present invention provide an alternative to site-directed mutagenesis and chemical modification for introducing unnatural amino acids into proteins. Moreover, the methods of the present invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure-activity relationships (xe2x80x9cSARsxe2x80x9d)) of non-natural variants of the proteins.
An advantage of the present invention is that it is possible to introduce predetermined glycosylation patterns into proteins in a simple and repeatable manner. This advantage provides an ability to modify critical protein characteristics such as partitioning, solubility, cell-cell signaling, catalytic activity, biological activity and pharmacological activity. Additionally, the methods of the present invention provide for a mechanism of xe2x80x9cmaskingxe2x80x9d certain chemically or biologically important protein sites, for example, sites which are critical for immunological or allergenic response or sites which are critical to proteolytic degradation of the modified protein.
Another advantage of the present invention is the ability to glycosylate a protein which is not generally glycosylated, or to modify the glycosylation pattern of a protein which is generally glycosylated.
Another advantage of the present invention is to produce enzymes which have altered catalytic activity. In one specific example, the inventors herein have shown that it is possible to modify the substrate specificity of a protease to increase the esterase activity as compared to the amidase activity. Similarly, modifications of substrate specificity would be expected when utilizing the present invention with other enzymes.
These and other advantages of the present invention are described in more detail in the following detailed description.